Apoptosis, or programmed cell death, is a crucial life-cycle decision point for cells in multicellular organisms from C. elegans to humans. It is a vital process in normal embryogenesis and development, maintenance of homeostasis, and immune system function. The regulation and initiation of apoptosis is a complex and intricately regulated process, consistent with this diversity of function. The death domain superfamily has emerged as the prime mediator of the interactions necessary for transducing a death signal. This superfamily consists of death domain (DD), death effector domain (DED) and caspase recruitment domain (CARD) families. Each of these families interacts with other proteins through homotypic interactions in which CARD-CARD, DD-DD and DED-DED contacts are formed exclusively.
Caspases are the primary executioners of apoptosis, with effector caspases cleaving essential proteins such as poly(ADP-ribose) polymerase (PARP), and activating endonucleases such as CAD (by cleavage of the inhibitor ICAD). Upstream caspases, such as Caspases 8 and 9, are activated by signaling complexes such as the death-inducing signaling complex (DISC) and the apoptosome, respectively. Binding of caspases to specific adaptor molecules via CARD or DED domains leads to autoactivation of caspases. For example, the apoptosome consists of Apaf-1, Caspase 9 and cytochrome c and, within this structure, Apaf-1 interacts with proCaspase 9 via a CARD-CARD interaction. Cytochrome c binding to Apaf-1 activates the complex to allow recruitment and autoactivation of proCaspase 9. It is probable that the complex contains multiple Apaf-1 and proCaspase-9 molecules as the active complex has a molecular weight of 700 kDa.
The apoptosome, as well as the Fas DISC and the Pelle-containing complex are examples of large regulatory complexes involved in apoptosis. The proteins in these complexes are composed of multiple domains such as protein-protein interaction motifs, kinase domains, proteolytic domains, ligand-binding domains and membrane-binding domains. The death domains invariably provide one of the means for intramolecular communication. For a review, see C. H. Weber and C. Vincenz, “The death domain superfamily: a tale of two interfaces?” Trends in Biochemical Sciences, Vol. 26 No. 8, 2001.
Caspase 9 is a member of the aspartate-specific cysteine protease (ASCP) family of proteases that includes, for example, ICE, CPP32, Nedd2/Ich-1, Mch2, Mch3, Mch4, Mch5, TX (ICH-2, ICErel-II), and ICErel-III.
Caspase 9 shares amino acid sequence homology with several ASCPs, but its catalytic site QACGG differs in the fourth residue from the relatively conserved catalytic sites in other known ACSPs. U.S. Pat. No. 6,271,361, which discloses the DNA and amino acid sequences for Caspase 9, is hereby incorporated by reference in its entirety.
Like many ASCPs, Caspase 9 is synthesized as a proenzyme, which can be proteolytically cleaved by, for example, CPP32 or granzyme B. Cleavage of Caspase 9 yields two subunits, a large subunit and a small subunit, which associate to form an active heterodimer complex. In particular, CPP32 can cleave proCaspase 9 into a large subunit having an approximate molecular weight of 37 kDa (p37) and a small subunit having an approximate molecular weight of 10 kDa (p10). Similarly, granzyme B can cleave proCaspase 9 into a large subunit having an approximate molecular weight of 35 kDa (p35) and a small subunit having an approximate molecular weight of 12 kDa (p12). Moreover, other components of the apoptotic pathway can process Caspase 9 into a larger and a smaller cleavage product. Accordingly, the terms “large subunit” and “small subunit” will readily be understood to refer to any larger proteolytic cleavage product such as p37 or p35, and any smaller cleavage product such as p10 or p12, respectively.
Like other ASCPs, the active Caspase 9 complex can act as a protease and requires an Asp residue in the P1 position of the substrate binding site with a small, preferably hydrophobic, residue in the P1′ position.
Apoptosis plays a significant role in numerous pathological conditions in that programmed cell death is either inhibited, resulting in increased cell survival, or enhanced, which results in the loss of cell viability. Examples of pathological conditions resulting from increased cell survival include cancers such as lymphomas, carcinomas and hormone-dependent tumors. Such hormone-dependent tumors include, for example, breast, prostate and ovarian cancer. Increased cell survival or apoptosis inhibition can also result in autoimmune diseases such as systemic lupus erythematosus and immune-mediated glomerulonephritis, as well as viral infections such as herpesvirus, poxvirus and adenovirus. The first gene identified as being involved in a cell death pathway, the bcl-2 gene, was identified in cancer cells and was shown to function by decreasing the likelihood that cells expressing the gene would undergo apoptosis.
In contrast, apoptotic diseases where enhanced programmed cell death is a prevalent cause generally includes, for example, degenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration, and the encephalopathy associated with acquired immunodeficiency disease (AIDS). Since nerve cells generally do not divide in adults and, therefore, new cells are not available to replace the dying cells, the nerve cell death occurring in such diseases results in the progressively deteriorating condition of patients suffering from the disease. Other diseases associated with increased apoptosis include, for example, myelodysplastic syndromes such as aplastic anemia and ischemic injury, including myocardial infarction, stroke and reperfusion injury.
Caspase 9 inhibitors include those that inhibit protease activity as well as compounds that inhibit Caspase 9 binding to other polypeptides. Such compounds are useful as pharmaceuticals for treating or preventing diseases characterized by apoptotic cell death. When used in the present invention Caspase 9 polypeptides can be used to screen for compounds that activate or act as agonists of Caspase 9, such as by inducing cleavage of the proenzyme into its active subunits. Such compounds are similarly useful as pharmaceuticals for treating or preventing diseases characterized by the loss of apoptotic cell death.